The present invention relates to polypeptides which are derivatives of a staphylococcal protein A (SPA) domain, more specifically the B or Z domain, wherein between 1 and 20 amino acid residues of the said SPA domain have been substituted by other amino acid residues, said substitution resulting in interaction capacity of the said polypeptide with human Factor VIII protein. The said polypeptides are useful e.g. in the purification of Factor VIII protein and in diagnosis of hemophilia.
Random mutagenesis in combination with an efficient phenotypic selection procedure has proved to be an important tool in molecular biology to analyze the structure and function of proteins. Interesting targets for random mutagenesis followed by phenotypic selection are solvent-exposed surfaces of bacterial receptors. Such proteins can be unusually stable, which makes them suitable for various applications (Alexander et al. (1992) Biochemistry 31, 3597-3603). In particular, for bacterial receptors containing helix bundle structures, the conformation can be expected to be tolerant to changes in the side chains of residues not involved in helix packing interfaces. Examples of such molecules are the relatively small (58 residues) IgG-binding domain B of staphylococcal protein A (SPA) and the synthetic analogue of domain B, designated domain Z (Nilsson et al. (1987) Protein Engineering 1, 107-113).
The SPA-derived domain Z (SEQ ID NO: 1) has been utilized as a scaffold for constructing domain variants with new functions. Repertoires of mutant Z domain genes were assembled and inserted into a phagemid vector adapted for monovalent phage display of Z domain variants. Two combinatorial libraries, each comprising approximately 4xc3x97107 transformants, were constructed. Selection against different target proteins, viz. Taq DNA polymerase, human insulin and a human apolipoprotein A-1 variant, was performed. The obtained binding proteins were referred to as xe2x80x9caffibodiesxe2x80x9d. See WO 95/19374; Nord et al. (1995) Protein Engineering, Vol. 8 (6), 601-608 (hereinafter referred to as Nord-95); and Nord et al. (1997) Nature Biotechnology, Vol. 15, 772-777 (hereinafter referred to as Nord-97).
Hemophilia is an inherited disease which has been known for centuries, but it is only within the last four decades that it has been possible to differentiate between the various forms; hemophilia A and hemophilia B. Hemophilia A is the most frequent form. It affects only males with an incidence of one or two individuals per 10,000 liveborn males. The disease is caused by a strongly decreased level or absence of biologically active coagulation Factor VIII (also known as antihemophilic factor, AHF), which is a protein normally present in plasma.
Therapeutic Factor VIII concentrates for the treatment of hemophilia have been prepared by fractionation of plasma. Factor VIII concentrates derived from human plasma contain several fragmented fully active Factor VIII forms as described by Andersson et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2979-2983. The smallest active form hitherto described has a molecular mass of 170 kDa and consists of two chains of 90 kDa and 80 kDa, respectively, held together by metal ion(s). Reference is here made to EP-A-0 197 901.
However, methods are also available for production of Factor VIII in cell culture using recombinant DNA techniques, as reported e.g. by Wood et al. (1984) Nature 312, 330-337 and in EP-A-0 160 457. The structure and biochemistry of recombinant Factor VIII products in general have been described by Kaufman, R. J. in Trends in Biotechnology Vol. 9(10), 353-359, 1991; and in Hematology Vol. 63, 155-165, 1991. A recombinant Factor VIII form, termed r-VIII SQ, which corresponds to the 170 kDa plasma form, is described in WO 91/09122.
There is a need for new polypeptides which have interaction capacity with the Factor VIII protein and which can be used e.g. for purification of Factor VIII, for diagnosis of conditions related to Factor VIII deficiency, such as hemophilia, in therapy, or for research purposes.